Genotyping kit for diagnosis of human papilloma virus infection

ABSTRACT

The present invention relates to a genotyping kit for diagnosis of detecting the human papillomavirus (HPV) infection, probes for genotyping the HPV, and DNA chips including the probes. Also, the present invention relates to a method for diagnosis of HPV infection.

BACKGROUND OF THE INVENTION

[0001] (a) Field of the Invention

[0002] The present invention relates to a genotyping kit and method fordiagnosis of human papillomavirus (HPV) infection, probes for genotypingthe HPV, and DNA chips including the probes. More specifically, thepresent invention relates to a genotyping kit for detecting humanpapillomaviruses from clinical samples of infected patients using a DNAchip, a process for preparing the said DNA chip, and a method fordiagnosis of HPV infection using the genotyping kit.

[0003] (b) Description of the Related Art

[0004] Uterine cancer includes cervical cancer, endometrial cancer,uterine sarcoma and the like. For cervical cancer, approximately 450,000new cases occur worldwide each year, approximately 6,000 of these inKorea. Since the occurrence of cervical cancer (including cervicalintraepithelial neoplasia) accounts for 22.1% of total cancer cases inKorean women, the highest incidence with the second highest death rate,the prevention, diagnosis and treatment of cervical cancer are regardedas the most important issues in women's health.

[0005] Cervical cancer progresses through a precancerous stage, cervicalintraepithelial neoplasia (CIN), known to be mainly caused by humanpapillomavirus (HPV) infection. Especially, infection by particulartypes of HPV raises the possibility of developing invasive disease. Over70 genotypes of HPV have been identified since the recognition of HPV asthe main etiological factor for cervical cancer, and through research,certain HPV genotypes have been selectively found in lesions of specificlocations or progression stages, which has furthered recognition of thebiological diversity of HPV infection. Among the. HPV genotypes detectedin the anogenital area, over 10 genotypes have been classified as beingpart of the high-risk group that are associated with an elevated riskfor developing cervical cancer. Based on these findings,characterization of the biological differences of HPV infection isconsidered to be of significant importance to the diagnosis andprevention of cervical cancer.

[0006] The test that has been most commonly used for the diagnosis ofcervical cancer at its early stage is the Pap smear, which is acytological test performed as follows: old cells removed from theoutermost layer of cells from the surface of the cervix are stained andexamined for histopathological characteristics of HPV infectionincluding koilocytosis, perinuclear halo formation in the epithelialcells. However, due to the low diagnostic efficiency (1-15%) of Paptests together with other limitations, additional methods such ascolposcopy are necessary for more dependable diagnoses. Colposcopicscreening can detect HPV infection at up to 70%, but it hasdisadvantages including high cost of the equipment, the need for skilledinterpreters, and inability of determining HPV genotypes to distinguishbetween high-risk and low-risk infection. Therefore, efforts have beencontinuously made to develop techniques for the detection of HPV andidentification of HPV genotypes, to supplement conventional screeningmethods for cervical cancer and its precursors such as the Pap test.

[0007] The methods for detection of HPV and identification of HPVgenotypes can be classified into two groups, i.e., direct detection ofHPV DNA and detection of amplified HPV DNA. The methods for directdetection of HPV DNA include liquid hybridization (Hybrid Capture kit byDigene Diagnostics, Silver Spring, Md., USA, www.digene.com), Southernblot and dot blot with HPV type-specific probes, filter in situhybridization (FISH) and the like; and methods for the detection ofamplified DNA include type-specific PCR (polymerase chain reaction) andgeneral-primer PCR. In particular, genotype analyses of amplified HPVDNA by general primer sets are commonly performed by employing dot blothybridization, microtitre plate hybridization, or a line probe assay.Among these methods, liquid hybridization by Hybrid Capture and lineprobe assay following a general-primer PCR have been considered mostsuitable for diagnostic purposes. The line probe assay can detect about20 different HPV genotypes by immobilizing oligonucleotide probes on anitrocellulose membrane, however it lacks reliability due to lowsensitivity and difficulties in data interpretation. CommercializedHybrid Capture kits can detect HPV DNA in clinical samples without PCRamplification, and they can distinguish between high-risk and low-riskHPV groups. But the fact that Hybrid Capture kits cannot identify thegenotypes of infecting HPV limits accurate risk determination since therisk factors amongst high-risk HPVs are not the same, in other words,intermediate-risk types are included in the high-risk group. Moreover,the use of an RNA probe may present low stability of the kit, and thepossibility of contamination cannot be excluded.

[0008] Under these circumstances, there have been strong reasons forexploring and developing a simple and accurate method and a genotypingkit for detection of HPV infection and identification of the genotype ofinfecting HPV with high specificity and sensitivity, as well as probesfor genotyping the HPV, and DNA chips including the probes. In addition,the genotyping kit, the probes and the DNA chips must have specificityand sensitivity high enough to detect various kinds of HPV so that theycan diagnose the HPV infection and identify its genotype with accuracy.

SUMMARY OF THE INVENTION

[0009] It is one object of the present invention to provide novel probeshaving nucleotide sequences complementary to DNA of HPV in order todetect HPV infection and identify the genotype of HPV with highspecificity and sensitivity.

[0010] It is another object of the invention to provide a DNA chip fordetection of HPV infection and identification of the HPV genotype,wherein the DNA chip comprises probes having nucleotide sequencescomplementary to DNA of HPV.

[0011] It is yet another object to provide an HPV genotyping kitcomprising a novel DNA chip for detection of HPV infection andidentification of the HPV genotype.

[0012] It is still another object to provide a method for diagnosis ofHPV infection and identification of the HPV genotype using a novel DNAchip for detection of HPV infection and identification of the HPVgenotype.

[0013] It is a further object to provide a method for amplifying sampleDNA by a novel PCR in the diagnosis of HPV infection and theidentification of the HPV genotype using a novel DNA chip for detectionof HPV infection and identification of the HPV genotype.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIGS. 1a and 1 b are schematic representations of the types andpositions of the probes on the DNA chip according to one embodiment ofthe present invention.

[0015]FIG. 2a is a photograph showing the result of HPV 16 DNA analysisusing the DNA chip shown in FIG. 1a.

[0016]FIG. 2b is a photograph showing the result of HPV 18 DNA analysisusing the DNA chip shown in FIG. 1a.

[0017]FIG. 2c is a photograph showing the result of HPV 39 DNA analysisusing the DNA chip shown in FIG. 1a.

[0018]FIG. 2d is a photograph showing the result of HPV 58 DNA analysisusing the DNA chip shown in FIG. 1a.

[0019]FIG. 2e is a photograph showing the result of HPV 68 DNA analysisusing the DNA chip shown in FIG. 1a.

[0020]FIG. 2f is a photograph showing the result of HPV 69 DNA analysisusing the DNA chip shown in FIG. 1a.

[0021]FIG. 2g is a photograph showing the result of HPV 6 DNA analysisusing the DNA chip shown in FIG. 1a.

[0022]FIG. 2h is a photograph showing the result of HPV 11 DNA analysisusing the DNA chip shown in FIG. 1a.

[0023]FIG. 2i is a photograph showing the result of HPV 43 DNA analysisusing the DNA chip shown in FIG. 1a.

[0024]FIG. 2j is a photograph showing the result of HPV 44 DNA analysisusing the DNA chip shown in FIG. 1a.

[0025]FIG. 2k is a photograph showing the result of HPV 61 DNA analysisusing the DNA chip shown in FIG. 1b.

[0026]FIG. 2l is a photograph showing the result of HPV 67 DNA analysisusing the DNA chip shown in FIG. 1b.

[0027]FIG. 3a is a photograph showing the result of analyzing a sampleinfected with HPV 16 using the DNA chip of the present invention.

[0028]FIG. 3b is a photograph showing the result of analyzing a sampleinfected with HPV 31 using the DNA chip of the present invention.

[0029]FIG. 3c is a photograph showing the result of analyzing a sampleinfected with HPV 51 using the DNA chip of the present invention.

[0030]FIG. 3d is a photograph showing the result of analyzing a sampleinfected with HPV 52 using the DNA chip of the present invention.

[0031]FIG. 3e is a photograph showing the result of analyzing a sampleinfected with HPV 56 using the DNA chip of the present invention.

[0032]FIG. 3f is a photograph showing the result of analyzing a sampledouble-infected with HPV 16 and HPV 68 using the DNA chip of the presentinvention.

[0033]FIG. 3g is a photograph showing the result of analyzing a sampledouble-infected with HPV 16 and HPV 58 using the DNA chip of the presentinvention.

[0034]FIG. 3h is a photograph showing the result of analyzing a sampledouble-infected with HPV 33 and HPV 56 using the DNA chip of the presentinvention.

[0035]FIG. 3i is a photograph showing the result of analyzing a sampletriple-infected with HPV 16, HPV 52 and HPV 59 using the DNA chip of thepresent invention.

[0036]FIG. 3j is a photograph showing the result of analyzing a sampletriple-infected with HPV 16, HPV 35 and HPV 59 using the DNA chip of thepresent invention.

[0037]FIG. 4 is a photograph showing the result of DNA amplification bytwo-step PCR according to one preferred embodiment of the presentinvention.

[0038]FIG. 5 is a photograph showing the result of DNA amplification bymultiplex PCR according to one preferred embodiment of the presentinvention.

[0039]FIG. 6 is a schematic representation of the types and positions ofthe probes of the present invention and the probes of prior art.

[0040]FIG. 7a is a photograph showing the result of HPV 16 DNA analysisusing the DNA chip shown in FIG. 6.

[0041]FIG. 7b is a photograph showing the result of HPV 18 DNA analysisusing the DNA chip shown in FIG. 6.

[0042]FIG. 7c is a photograph showing the result of HPV 56 DNA analysisusing the DNA chip shown in FIG. 6.

[0043]FIG. 7d is a photograph showing the result of HPV 43 DNA analysisusing the DNA chip shown in FIG. 6.

[0044]FIG. 7e is a photograph showing the result of HPV 68 DNA analysisusing the DNA chip shown in FIG. 6.

[0045]FIG. 8 is a photograph showing the result of HPV 66 DNA analysisusing the HPV 66 probes of the present invention and the HPV 66 probesof the prior art.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] The genotyping kit of the invention for diagnosis of humanpapillomavirus (HPV) infection comprises: a DNA chip with probes thathave nucleotide sequences complementary to DNA of HPV; primers foramplifying DNA obtained from clinical samples by PCR; and means forlabeling amplified DNA hybridized with the probes of the said DNA chip.

[0047] The present inventors have tried to detect HPV infection andidentify the types of HPV by way of genotyping DNA from clinical samplesand prepared probes that have nucleotide sequences complementary to theDNA of HPV, and a DNA chip including the said probes. Further, weprepared an HPV genotyping kit comprising a DNA chip including probesthat have nucleotide sequences complementary to the DNA of HPV, primersfor amplifying DNA obtained from clinical samples by PCR, and means forlabeling amplified DNA hybridized to the probes of the said DNA chip,and we successfully detected HPV infection and identified genotypes ofinfecting HPV with the aid of the genotyping kit in a simple andaccurate manner.

[0048] The present invention provides nucleotide sequences of HPV DNA,preferably nucleotide sequences complementary to the L1 region of HPVDNA as probes. The probes of the present invention comprise nucleotidesequences set forth in SEQ ID NO: 1-30 (Tables 1a and 1b). In Table 1,the underlined regions on SEQ ID NO: 25 are additional sequences toprovide the conformation suitable for the function of probes. TABLE 1aProbes for newly added HPV Types SEQ ID NO. HPV Type Sequence 1 135′-ACATCATCTCTTTCAGACACATATAAGGCC-3′ 2 265′-AGTACATTATCTGCAGCATCTGCATCCACT-3′ 3 305′-TTATCCACATATAATTCAAGCCAAATTAAA-3′ 4 435′-CCTCTACTGACCCTACTGTGCCCAGTACAT-3′ 5 535′-TCTACATATAATTCAAAGCAAATTAAACAGTA-3′ 6 545′-TACAGCATCCACGCAGGATAGCTTTAATAAT-3′ 7 545′-CATCCACGCAGGATAGCTTTAATAATTCTG-3′ 8 555′-TGTGCTGCTACAACTCAGTCTCCATCTACAACA-3′ 9 575′-GAAACTAATTATAAAGCCTCCAATTATAAGGAA-3′ 10 615′-CTGTATCTGAATATAAAGCCACAAGCTTTAG-3′ 11 615′-CCTGTATCTGAATATAAAGCCACAAGCTTT-3′ 12 625′-CCTCCACTGCTGCAGCAGAATACACGGCTA-3′ 13 645′-TACAAATCCACCATATGCAAACACTAATTTTAA-3′ 14 675′-TATGTTCTGAGGAAAAATCAGAGGCTACAT-3′ 15 685′-TTTGTCTACTACTACTGAATCAGCTGTACCAAA-3′ 16 695′-AATCTGCATCTGCCACTTTTAAACCATCAGATT-3′ 17 705′-AACGGCCATACCTGCTGTATATAGCCCTAC-3′ 18 705′-ACCGAAACGGCCATACCTGCTGTATATAGC-3′ 19 745′-CGCCTTCTGCTACATATAATAGTTCAGACT-3′ 20 JC97105′-AAACACCCTCTGACACATACAAGGCTTCCA-3′ 21 165′-GTGCTGCCATATCTACTTCAGAAACTACAT-3′ 22 165′-TATGTGCTGCCATATCTACTTCAGAAACTACATA-3′ 23 345′-GGTACACAATCCACAAGTACAACTGCACCA-3′ 24 355′-TTCTGCTGTGTCTTCTAGTGACAGTACATA-3′ 25 355′-GCACGGTCTGTGTGTTCTGCTGTGTCTTCTACGTGC-3′ 26 405′-CTTATGTGCTGCCACACAGTCCCCCACACC-3′ 27 565′-TATTAGTACTGCTACAGAACAGTTAAGTAA-3′ 28 585′-CACTGAAGTAACTAAGGAAGGTACATATAA-3′ 29 595′-TCTACTACTTCTTCTATTCCTAATGTATAC-3′ 30 665′-CTAAAAGCACATTAACTAAATATGATGCCC-3′

[0049] TABLE 1b 31  6 5′-ATCCGTAACTACATCTTCCACATACACCAA-3′ 32 115′-ATCTGTGTCTAAATCTGCTACATACACTAA-3′ 33 185′-TGCTTCTACACAGTCTCCTGTACCTGGGCA-3′ 34 315′-TGTTTGTGCTGCAATTGCAAACAGTGATAC-3′ 35 335′-TTTATGCACACAAGTAACTAGTGACAGTAC-3′ 36 395′-TCTACCTCTATAGAGTCTTCCATACCTTCT-3′ 37 425′-CTGCAACATCTGGTGATACATATACAGCTG-3′ 38 445′-GCCACTACACAGTCCCCTCCGTCTACATAT-3′ 39 455′-ACACAAAATCCTGTGCCAAGTACATATGAC-3′ 40 515′-AGCACTGCCACTGCTGCGGTTTCCCCAACA-3′ 41 525′-TGCTGAGGTTAAAAAGGAAAGCACATATAA-3′ 42 β-globin5′-TGCACCTGACTCCTGAGGAGAAGTCTGCCG-3′ 47 165′-GTCATTATGTGCTGCCATATCTACTTCAGA-3′ 48 345′-TACACAATCCACAAGTACAAATGCACCATA-3′ 49 355′-GTCTGTGTGTTCTGCTGTGTCTTCTAGTGA-3′ 50 405′-GCTGCCACACAGTCCCCCACACCAACCCCA-3′ 51 565′-GTACTGCTACAGAACAGTTAAGTAAATATG-3′ 52 585′-ATTATGCACTGAAGTAACTAAGGAAGGTAC-3′ 53 595′-CTGTGTGTGCTTCTACTACTGCTTCTATTC-3′ 54 665′-CTATTAATGCAGCTAAAAGCACATTAACTA-3′

[0050] The probes of the present invention enable highly specific andsensitive detection of HPV infection and identification of the HPVgenotype, and have higher specificity and sensitivity than the probesfor HPV genotyping described in PCT/KR/01213 (filed on Oct. 26, 2000).Among the probes of the present invention, the probes set forth in SEQID NO: 1-20, HPV 13, 26, 30, 43, 53, 54, 55, 57, 61, 62, 64, 67, 68, 69,70, 74, and JC9710 are not included in the genotyping kit described inthe above application, and therefore the probes according to the presentinvention enable it to be an implement that can detect more varioustypes of HPV, contributing to its accuracy. The probes for HPVgenotyping described in PCT/KR/01213 further comprise additional probesfor HPV 16, 34, 35, 40, 56, 58, 59, and 66, along with nucleotidesequences set forth in SEQ ID NO: 31-41.

[0051] The DNA chip of the present invention may comprise one or moreHPV probes selected from nucleotide sequences set forth in SEQ ID NO:1-30. In addition, it may further comprise a β-globin sequence as amarker, and preferably the nucleotide sequence set forth in SEQ ID NO:42. In one embodiment of the invention, the DNA chip comprises DNA chipsincluding all probe sequence sets that are easily combined by thoseskilled in the art. It includes the following.

Embodiment 1

[0052] A DNA chip comprising one or more HPV probes selected from thegroup consisting of oligonucleotides having nucleotide sequences setforth in SEQ ID NO: 1 to 30 and oligonucleotides having nucleotidesequences complementary to the said oligonucleotides.

Embodiment 2

[0053] A DNA chip comprising:

[0054] (i) one or more HPV probes selected from the group consisting ofoligonucleotides having nucleotide sequences set forth in SEQ ID NO: 1to 30 and oligonucleotides having nucleotide sequences complementary tothe said oligonucleotides; and

[0055] (ii) one or more HPV probes selected from the group consisting ofprobes having nucleotide sequences set forth in SEQ ID NO: 31 to 41 andnucleotide sequences complementary to the said nucleotide sequences.

Embodiment 3

[0056] A DNA chip comprising:

[0057] (i) one or more HPV probes selected from the group consisting ofprobes having nucleotide sequences set forth in SEQ ID NO: 1 to 20 andnucleotide sequences complementary to the said nucleotide sequences;

[0058] (ii) 8 HPV probes selected from the group consisting of probeshaving nucleotide sequences set forth in SEQ ID NO: 21 to 30 andnucleotide sequences complementary to the said nucleotide sequences; and

[0059] (iii) 11 HPV probes having nucleotide sequences set forth in SEQID NO: 31 to 41 and nucleotide sequences complementary to the saidnucleotide sequences.

Embodiment 4

[0060] A DNA chip comprising:

[0061] (i) 9 HPV probes selected from the group consisting of probeshaving nucleotide sequences set forth in SEQ ID NO: 4 to 10;

[0062] (ii) 8 HPV probes selected from the group consisting of probeshaving nucleotide sequences set forth in SEQ ID NO: 21 to 30 andnucleotide sequences complementary to the said nucleotide sequences; and

[0063] (iii) 11 HPV probes having nucleotide sequences set forth in SEQID NO: 31 to 41 and nucleotide sequences complementary to the saidnucleotide sequences.

Embodiment 5

[0064] A DNA chip comprising:

[0065] (i) 3 HPV probes having nucleotide sequences set forth in SEQ IDNO: 4, 15, and 16;

[0066] (ii) 8 HPV probes selected from the group consisting of probeshaving nucleotide sequences set forth in SEQ ID NO: 21 to 30 andnucleotide sequences complementary to the said nucleotide sequences; and

[0067] (iii) 11 HPV probes having nucleotide sequences set forth in SEQID NO: 31 to 41 and nucleotide sequences complementary to the saidnucleotide sequences.

[0068] In one embodiment of the invention, the DNA chip may furthercomprise position markers to locate probes. The position markersinclude, but are not limited to, β-globin, actin, and GAPDH(glyceraldehyde-3-phosphate dehydrogenase) genes, and the like. Thepreferable position marker is β-globin, whose sequence is set forth inSEQ ID NO: 42.

[0069] According to the present invention, probes that have nucleotidescomplementary to DNA of HPV are provided. The probes of the inventionare characterized to specifically hybridize with DNA of HPV.

[0070] The process for preparing a DNA chip contained in the said HPVgenotyping kit comprises the steps of: preparing 5′ terminalamine-linked DNA probes which have nucleotide sequences complementary toDNA of HPV; affixing the DNA probes thus prepared to analdehyde-derivatized solid surface; and reducing excessive aldehydes notreacted with amine. The process for preparing a DNA chip of theinvention is described in more detail by the following steps.

[0071] Step 1: Preparation of Probes

[0072] 5′ terminal amine-linked DNA probes that have nucleotidesequences complementary to the DNA of HPV are prepared. The nucleotidesequences of the probes are designed and synthesized to have nucleotidesequences complementary to the DNA of HPV, preferably the L1 region ofHPV DNA, and the probes are prepared by linking an amine group at the 5′terminal of the nucleotide sequences which enables the probes to bind toan aldehyde-derivatized solid surface.

[0073] Step 2: Affixture of Probes

[0074] DNA probes prepared in Step 1 are affixed to analdehyde-derivatized surface of a solid support, preferably glass. Theprobes are affixed to the surface of the solid support via a Schiff'sbase reaction between an aldehyde group on the surface of the solidsupport and an amine group at the 5′ terminal of the probe under thecondition of a 30 to 40° C. temperature and 70 to 100% humidity, whilecontrolling the concentration of probes in a range of preferably 100 to300 pmol/μl, and more preferably 200 pmol/μl. Also, β-globin whichfunctions as a control for position markers and the hybridizationreaction are affixed to the surface of the solid support.

[0075] Step 3: Preparation of DNA Chip

[0076] Excessive aldehydes not reacted with the amine on the solidsurface are reduced by employing a reducing agent of NaBH₄, to finallyprepare a DNA chip.

[0077] The present invention also provides an HPV genotyping kitcomprising the said DNA chip and a method for diagnosis of HPV infectionusing the said genotyping kit. The present invention relates to an HPVgenotyping kit comprising:

[0078] (i) a DNA chip according to the invention;

[0079] (ii) primers for amplifying DNA obtained from clinical samples byPCR; and,

[0080] (iii) means for labeling amplified DNA hybridized with the saidDNA chip.

[0081] The present invention also relates to a method for diagnosis ofHPV infection comprising the steps of:

[0082] (i) amplifying DNA obtained from clinical samples by PCR withprimers of the HPV genotyping kit, for example primers set forth in SEQID NO: 43 and 44;

[0083] (ii) applying the amplified DNA to the DNA chip of the HPVgenotyping kit to hybridize the amplified DNA with probes of the DNAchip; and

[0084] (iii) detecting DNA bound on the surface of the DNA chip afterlabeling hybridized DNA.

[0085] In one embodiment according to the invention, amplification ofsample DNA may be carried out via a usual PCR, preferably a two-step PCRor a multiplex PCR that amplifies using more than 2 primers at the sametime, and more preferably a multiplex two-step PCR that amplifies in twosteps using more than 2 primers.

[0086] The development of the best condition for the HPV DNA sequenceamplification methodology resulted in the two-step PCR or the multiplexPCR.

[0087] PCR consists of pre-denaturation, denaturation, primer annealing,and chain extension.

[0088] While conventional PCR is performed by repeating one step underthe same conditions, two-step PCR is performed with anotheramplification step, that is, two-step PCR is performed by repeating oneor more steps among denaturation, primer annealing, or chain extensionunder different conditions. According to the present invention, thefirst amplification step is carried out for a shorter time than that ofthe conventional chain extension step, and the second amplification stepis carried out for an even shorter time than that of the firstamplification step. Therefore, it prevents obtaining lengthy productswhen the extension reaction is carried out-for a longer time (e.g. 1 to2 minutes), and thus increases the specificity of probes, which ispreferable. More preferably, a chain extension of the PCR amplificationmay be carried out for 20 to 30 seconds in the first step, and for 10 to15 seconds in the second step.

[0089] In the present invention, while it is possible to amplify the HPVprimer sample, the β-globin sample, and a marker separately, one-stepamplification using HPV primers set forth in SEQ ID NO: 43-44 andβ-globin primers set forth in SEQ ID NO: 45-46 can be performedtogether. Otherwise, two-step PCR as mentioned above can also be done.

[0090] PCR amplification may be carried out using an HPV primer sampleand a β-globin sample, respectively, or it may be carried out in onestep or two steps using HPV primers set forth in SEQ ID NO: 43-44 andβ-globin primers set forth in SEQ ID NO: 45-46, concomitantly.

[0091] In one preferred embodiment of the invention, PCR was performedin a PCR thermocycler containing the said primers and β-globin, dATP,dCTP, dGTP, dTTP, labeling materials, and Taq polymerase, and optionallyBSA and DMSO, with 5 cycles of denaturation for 5 min at 94° C.,denaturation for 1 min at 94° C., primer annealing for 2 min at 45˜50°C., and extension for 20˜30 sec at 72° C., followed by 35 cycles ofdenaturation for 1 min at 94° C., primer annealing for 2 min at 45˜50°C. and extension for 10˜15 sec at 72° C., and then further extension for2 min at 72° C., resulting in obtaining labeled amplified DNA samples.

[0092] The method for diagnosis of HPV infection using HPV genotypingkit of the invention is further illustrated by the following steps.

[0093] Step 1: Amplification of Sample DNA

[0094] DNA obtained from clinical samples is amplified using the primersof the HPV genotyping kit, where polymerase chain reaction (PCR)employing biotin-16-dUTP is carried out to give biotin-containingamplified DNA. 4 primers for amplification are set forth in Table 2:TABLE 2 SEQ ID HPV Base NO. Type Sequence Number 43 Gp5+5′-TTTKTTACHGTKGTDGATACYAC-3′ 23 44 Gp6+ 5′-GAAAHATAAAYTGYAADTCATAYTC-3′25 45 BG 1 5′-ATACAAGTCAGGGCAGAG-3′ 18 46 BG 25′-CTTAAACCTGTCTTGTAACC-3′ 20

[0095] In Table 1, R(A, g): Y(C, T): M(A, C): K(g, T): S(g, C): W(A, T),V(A, C, g): H(A, T, C): B(g, T, C): D(g, A, T): N(A, g, V, T), which iswell-known to those skilled in the art.

[0096] Primers for DNA amplifying composed of nucleotide sequences setforth in SEQ ID NO: 43 and 44 are provided, as well as novel β-globinprimers composed of nucleotide sequences set forth in SEQ ID NO: 45 and46.

[0097] Step 2: Hybridization

[0098] Amplified DNA thus obtained is applied to the DNA chip of the HPVgenotyping kit and hybridized with the probes of the DNA chip.

[0099] Step 3: Detection

[0100] The amplified sample DNA hybridized with the probes are labeledwith means for labeling and detected with a confocal laser scanner.

[0101] Means for labeling comprise, but are not limited to, Cy5,biotin-binding compound, Cy3, EDANS(5-(2′-aminoethyl)amino-1-naphthalene sulfuric acid),tetramethylrhodamine (TMR), tetramethylrhodamine isocyanate (TMRITC),x-rhodamine or Texas red. Preferably, Cy5 and biotin-binding compoundcan be used. Labeling materials are detected by a confocal laserscanner.

[0102] The HPV genotyping kit of the invention is an implement that candetect HPV infection in a simple and accurate manner, as well asidentify the types of infecting HPV, and therefore it may contribute toearly diagnosis, prevention, and treatment of cervical cancer.

[0103] The present invention is further shown in the following examples,which should not be taken to limit the scope of the invention.Particularly, though a DNA chip with 22 or 28 probes was prepared in theExamples described below, it is to be understood that the presentinvention is not limited by types and numbers of probes, but DNA chipsusing nucleotide sequences derived from HPV DNA and any variety ofdetection kits using the said DNA chips are intended to be includedwithin the scope of the invention.

EXAMPLE 1 Preparation of Probes

[0104] Prevalent HPV types including 15 high-risk types (HPV type 16,18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 69) and 7 low-risktypes (HPV type 6, 11, 34, 40, 42, 43, 44) were selected, andgenotype-specific probe β-globin for each HPV type possessing an aminegroup at the 5′ terminal of the sequence was prepared for the detectionof HPV genotypes. The nucleotide sequence of each probe is set forth inthe Sequence List.

EXAMPLE 2 Preparation of DNA Chip

[0105] A DNA chip was prepared as follows: each probe prepared inExample 1 was dissolved in 3×SSC (45 mM sodium citrate, 0.45M NaCl, pH7.0) at a concentration of 200 pmol/μl, and spotted onto analdehyde-derivatized silylated slide (CSS-100, CEL, Houston, Tex., USA)to form an array of spots with a size of 150 μm at 300 μm of spacingbetween spots using a microarrayer (GMS 417 Arrayer, TaKaRa, Japan),followed by performing a Schff's base reaction under the condition of a37° C. temperature and over 70% humidity, for 4 hours. The slide waswashed with 0.2% (w/v) sodium dodecyl sulfate (SDS), and withtriple-distilled water. Then the slide was treated with a NaBH₄ solution(0.1 g NaBH₄, 30ml phosphate buffered saline (PBS), 10 ml ethanol) for 5minutes to reduce excessive aldehydes not reacted with amine, followedby washing with triple-distilled water and air-drying. Also, β-globin,which functions as a control group for position markers and thehybridization reaction, is affixed to the surface of the solid support.

EXAMPLE 3 Optimal PCR Amplification

[0106] To establish multiplex PCR and amplification of HPV genes fromclinical samples, an Optimal HPV PCR was carried out under the followingconditions:

[0107] 3-1: Two-Step PCR Amplification

[0108] Method 1

[0109] To obtain Cys-labeled amplified DNA samples, PCR was performedwith 5 cycles of denaturation for 5 min at 94° C., denaturation for 1min at 94° C., primer annealing for 2 min at 50° C., and extension for30 sec at 72° C., followed by 35 cycles of denaturation for 1 min at 94°C., primer annealing for 2 min at 50° C., and extension for 15 sec at72° C., and then further extension for 2 min at 72° C.

[0110] Method 2

[0111] Cy5-linked amplified HPV DNA samples were prepared analogously asin Method 1, except for primer annealing for 30 sec at 50° C.

[0112] Method 3: Conventional One-Step PCR of Prior Art

[0113] Cy5-labeled amplified DNA was obtained and compared after 40cycles of denaturation for 5 min at 94° C., denaturation for 1 min at94° C., primer annealing for 2 min at 50° C., and extension for 30 secat 72° C., and then further extension for 2 min at 72° C.

[0114] When lanes 4˜6, lanes 8˜10, and lanes 11˜13 in FIG. 4 werecompared with one another, the conditions in methodology 2 (lanes 4, 8and 1 1) were proven to be the most specific and efficient.

[0115] 3-2: Multiplex PCR Amplification

[0116] The compositions and conditions for establishing multiplex PCRamplification of HPV are as follows.

[0117] In order to obtain Cy5-labeled amplified DNA samples, multiplexPCR using two-step PCR described in Example 3-1 was performed in athermocycler (Perkin-Elmer Cetus, Calif., USA) with 50 μl of reactionmixture containing a 2×PCR buffer (50 mM KCl, 4 mM MgCl₂; 10 mMTris-HCl; pH 8.3); 0.1 μg of DNA; 2˜4 mM MgCl₂; 25 pmol of HPV primersset forth in SEQ ID NO: 43-44; 5 pmol of β-globin primers set forth inSEQ ID NO: 44-45; 40 μM each of dATP, dCTP, dGTP (Pharmacia); 30 μM ofdTTP (Pharmacia); 0.5 μM Cy5-dUTP (NEN, USA); 2 units of Taq polymerase(TaKaRa, Japan); and optionally BSA and DMSO, with 5 cycles ofdenaturation for 5 min at 94° C., denaturation for 1 min at 94° C.,primer annealing for 2 min at 45-50° C., and extension for 20˜30 sec at72° C., followed by 35 cycles of denaturation for 1 min at 94° C.,primer annealing for 2 min at 45˜50° C. and extension for 10˜15 sec at72° C., and then further extension for 2 min at 72° C.

[0118] When lanes 2˜5 and lanes 6˜9 in FIG. 5 were compared, HPVamplification efficiencies of 1×PCR buffer monoplex PCR and of 2×PCRbuffer multiplex PCR were similar, and so the 2×PCR buffer multiplex PCRcondition was found to be the most specific and efficient.

EXAMPLE 4 Preparation of Samples

[0119] In order to detect HPV infection in human cervical swabs, DNA wasextracted from the said specimen and then purified. To test the adequacyof sample DNA, purified DNA was PCR amplified with β-globin primers setforth in SEQ ID NO: 45 and 46. The DNA samples which revealed β-globinDNA amplification were selected and used for further analyses of HPVDNA.

[0120] As HPV DNA standards, plasmid DNA comprising the HPV sequenceobtained from the following distributors were used: HPV types 6,11, 26,40, 45, 51 and 57 (Dr. Ethel-Michele de Villiers, AngewandteTumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld242, 69009 Heidelberg, Germany); HPV types 35, 44, and 56 (Dr. AttilaLörincz, Vice President, R&D and Scientific Director, DigeneDiagnostics, Inc., 2301-B Broadbirch Drive, Silver Spring, Md. 20904,USA); HPV types 42, 58, 59, 61, 64 and 67 (Dr. Toshihiko Matsukura,Department of Pathology and Laboratory of Pathology, AIDS ResearchCenter, National Institute of Infectious Disease, Tokyo 162, Japan); andHPV types 33, 34, 39, 52, 54, 66 and 70 (Dr. Gérard Orth, Unité MixteInstitut Pasteur/INSERM (U. 190), Institut Pasteur, 25 rue du DocteurRoux, 75724 Paris Cedex 15, France).

[0121] Additionally, DNA extracted and purified from the following celllines were used as positive controls: SiHa cell line (HPV 16, KCLB30035, Human squamous carcinoma, cervix), purchased from Korean CellLine Bank (Seoul National University, College of Medicine, Seoul,Korea).

[0122] The selected samples of DNA described above were PCR amplifiedusing primers set forth in SEQ ID NO: 43 and 44.

EXAMPLE 4-1 Preparation of Positive Control Samples

[0123] To obtain Cy5-labeled amplified DNA samples, HPV 16 and HPV 18DNA purified above were amplified by PCR with primers set forth in SEQID NO: 43 and 44. PCR was performed in a thermocycler (Perkin-ElmerCetus, Calif., USA) with 50 μl of a reaction mixture containing a 1×PCRbuffer (50 mM KCl, 4 mM MgCl₂, 10 mM Tris-HCl, pH 8.3); 0.1 μg of DNA; 4mM MgCl₂; 25 pmol of each primer, 40 μM each of dATP, dCTP, and dGTP(Pharmacia); 30 μM of dTTP (Pharmacia); 0.05 μM Cy5-dUTP (NEN, USA); and2 unit of Taq polymerase (TaKaRa, Japan) with 5 cycles of denaturationfor 5 min at 94° C., denaturation for 1 min at 94° C., primer annealingfor 2 min at 50° C., and extension for 30 sec at 72° C., followed by 35cycles of denaturation for 1 min at 94° C., primer annealing for 2 minat 50° C. and extension for 15 sec at 72° C., and then extension for 2min at 72° C.

EXAMPLE 4-2 Preparation of HPV Standards

[0124] Cy5-linked amplified HPV DNA samples were prepared analogously asin Example 4-1, except for employing templates of various HPV plasmidsdescribed above.

EXAMPLE 4-3 Preparation of Sample DNA from Clinical Samples

[0125] Cy5-linked amplified DNA samples were obtained analogously as inExample 4-1, except that DNA obtained from uterine cervical swabs wereused as templates, and primers set forth in SEQ ID NO: 43 and 44 wereemployed as primers.

EXAMPLE 5 Detection of HPV Infection Using DNA Chip

[0126] Amplified DNA samples obtained in Example 4 were applied to theDNA chip prepared in Example 2, and hybridization was carried out in ahybridization reaction chamber made up of a Cover slip (GRACE Bio-Labs,USA, PC4L-1.0) with a 100 μl capacity.

[0127] As for the quantity of hybridization reaction samples, 10 μl eachof amplified product was used for positive controls and plasmid DNA, anda mixture of 10 μl of HPV amplified product and 5 μl of beta-globinamplified product was used for DNA obtained from cervical swabs. Thesaid reaction samples were denatured by adding 3N NaOH solution (10%v/v) and standing them for 5 min at room temperature, and neutralized byadding 1 M Tris-HCl (pH 7.2, 5% v/v) followed by 3N HCl (10% v/v) andcooling them for 5 min on ice. The samples were then mixed with ahybridization solution made up of a 6×SSPE (saline-sodium phosphate-EDTAbuffer, Sigma Chemical Co., St. Louis, Mo., USA) and 0.2% SDS (sodiumdodecyl sulfate), and applied onto the DNA chip. The hybridizationreaction was carried out for 2 hours at 40° C., followed by washing witha 3×SSPE for 2 min and a 1×SSPE for 2 min, and air-drying at roomtemperature or spin-dryer drying.

[0128] The DNA chip hybridized with sample DNA was analyzed forfluorescent signals (extinction 650 nm, emission 668 nm) by using aconfocal laser scanner (GSI Lumonics, Germany) (see: FIG. 1, FIGS. 2a-2l and FIGS. 3a-3 j).

[0129]FIG. 1 is a schematic representation of the type and position ofthe probes on the DNA chip: each number indicates each HPV probe,circles(∘) indicate HPV and beta-globin probe-affixed positions, andclosed circles() indicate background markers to verify properperformance of the hybridization reaction and position markers forlocating probes. FIGS. 2a-2 e are photographs showing the results ofhigh-risk group HPV DNA analyses using HPV plasmids and cervical cancercell lines (HPV 16, 18, 39, 58 and 69), and FIGS. 2g-2 i are photographsshowing the results of low-risk group HPV DNA analyses using HPVplasmids (HPV 6, 11 and 40). As shown in FIGS. 2a-2 e and FIGS. 2g-2 i,hybridization signals produced by the amplified DNA of HPV plasmidstandards and HPV positive controls (cervical cancer cell lines) wereobserved clearly on the corresponding probes without significantcross-hybridization.

EXAMPLE 6 Detection of HPV Infection in Clinical Samples Using DNA Chip

[0130] In order to examine the accuracy and efficiency of diagnosis bythe DNA chip of the invention, clinical samples were PCR amplified withprimers comprising nucleotide sequences set forth in SEQ ID NO: 43-46,and then, for proper samples, a diagnostic procedure using the DNA chipwas performed to detect HPV infection as well as to determine the typeof infection.

[0131] DNA isolated from 213 uterine cervix specimens were amplifiedusing the method described in Example 4-3, and analyzed for HPVinfection by using the DNA chip of the invention. Detection of HPVincluded in the high-risk group was carried out using a Hybrid CaptureII method provided by the Digene Diagnostics company. The results of thetwo methods were compared (see: FIGS. 2a-2 l, FIGS. 3a-3 j, and Table3). FIGS. 2a-2 l and FIGS. 3a-3 j are photographs showing Examples ofthe results of DNA chip analyses of cervical swab specimens for HPVinfection. As shown in the Figures, detailed diagnoses of HPV infectionin the clinical samples via accurate detection and genotyping of theinfecting HPV were successfully accomplished by using the DNA chip ofthe invention. The results of the DNA chip analyses and Hybrid CaptureII analysis are shown in Table 3: TABLE 3 DNA Chip of the PresentInvention The Number of Single-infected Samples Hybrid HPV (The Numberof Percent Capture II Group Type Whole Infected samples) (%) PositiveHigh-risk HPV 16 31 *¹(66) 20.8 (44.3) 149 Group HPV 18  1 (18)  0.7(12.1) HPV 31  2 (3)  1.4 (2) HPV 33  2 (6)  1.4 (4) HPV 35  2 (6)  1.4(4) HPV 39  1 (3)  0.7 (2) HPV 45 — (1) — (0.7) HPV 51  6 (9)  4 (6) HPV52 10 (14)  6.7 (9.4) HPV 56 15 (27) 10.1 (18.1) HPV 58  7 (13)  4.7(8.7) HPV 59  2 (7)  1.4 (4.7) HPV 66  1  0.7 HPV 68  2 (6)  1.4 (4) HPV69 — — Low-risk HPV 6 — (1) — (0.7) Group HPV 11  1 (2)  0.7 (1.4) HPV34  1 (2)  0.7 (1.4) HPV 40  1 (2)  0.7 (1.4) HPV 42 — HPV 43  1  0.7HPV 44 — (1) — (0.7) Other type  4  2.7 Negative Negative HPV 18 51 50 1

[0132] In Table 1, “−” indicates absence of sample infected with HPV inthe corresponding item, and percent was calculated by {(the number ofsingle-infected samples or total number of infected samples)/149}×100.TABLE 4 Specificity and Sensitivity of DNA chip according to theinvention DNA Chip Hybrid Capture II Of The Invention Positive NegativeSum Positive 162  1* 163 Negative  0 50   50 Sum 162 51  213 RelativeSensitivity  100% Relative Specificity   98% (95% Cl = 94-100)Similarity 98.6% (95% Cl = 99-100) Copy Value(κ) 0.99 (95% Cl =0.96-1.00)

[0133] As shown in Table 4, DNA chip analysis according to the inventionwas reported to have 100% relative sensitivity and 98% relativespecificity as compared with the Hybrid Capture assay. The aboveinformation indicates that diagnosis of HPV infection using thegenotyping kit of the invention is superior in many aspects to theconventional methods employed for the same purpose.

[0134] In Tables 3 and 4, all samples which were confirmed as positiveby Hybrid Capture assay were tested or diagnosed with the DNA chip ofthe invention, and one of samples which were confirmed as positive byHybrid Capture assay was detected to be HPV 18 type. As a result ofsequencing to determine whether it was a false-positive, it wasconfirmed that it was HPV 18. While the Hybrid Capture assay can onlydiagnosis HPV infection, the DNA chip analysis can detect HPV infectionand identify the types of infecting HPV in a fast and accurate manner.The DNA chip analysis according to the invention is so highly sensitivethat it can detect the samples confirmed to be false-negative throughthe Hybrid Capture assay. As well-known to those skilled in the art,HIV16 type shows the highest frequency in HIV infection, followed by HIV56, 58 and 52 types in contrast to the Western tendency with HPV 18being the second frequent type.

[0135] As clearly illustrated and demonstrated above, the presentinvention provides a genotyping kit comprising said probes (SEQ ID NO: 1to 30) for identifying genotypes of HPV from clinical samples ofinfected patients and a method for diagnosis of HPV infection bygenotyping the infecting virus using the said genotyping kit.

COMPARATIVE EXAMPLE 1

[0136] As shown in Table 6, efficiency of the probes according to theinvention was compared with that of the conventional probes for HPV 16,34, 56, and 59, and a DNA chip to confirm efficiencies of additionalprobes for HPV 43, 68, and 69 was described. A test was carried outanalogously as in Example 6, except for employing the DNA chip describedabove.

[0137] As a result of analysis of control DNA for each type in FIGS.7-8, conventional probes were found to pose non-specificity (falsepositive) and low-sensitivity (false negative).

COMPARATIVE EXAMPLE 2 DNA Chip Test

[0138] A test was carried out analogously as in Example 6, except foremploying the conventional probes including probes set forth in SEQ IDNO: 31-41 and 47-54 and using the DNA chip of the invention. Sequencingwas performed using the conventional sequencing method.

[0139] Results of analysis using the conventional DNA chip art and thepresent DNA chip, results of sequencing via PCR for identification, andresults of analysis using the conventional probes and the present probesare shown in Tables 5a and 5b. It was found that in the frequency offalse negative and false positive, the genotyping kit using theconventional DNA chip is higher than the genotyping kit using the DNAchip of the invention.

[0140] The present invention provides a genotyping kit for diagnosingpatients infected with human papillomavirus (HPV), which includes thesaid probes, and a method for genotyping of HPV DNA isolated frompatients using the said genotyping kit in an accurate and fast manner.TABLE 5A Conventional DNA Chip Conventional HPV DNA Chip Sample HPV DNAChip Of The Invention Sequencing False Positive False Negative 1 OT 16,68 16, 68 16, 68 2 35 51 51 35 51 3 16 OT OT 16 4 59 59, 68 59, 68 68 540, 58 51 51 40, 58 51 6 16, 18, 33, 35 16, 18, 33 16, 18, 33 35 7 3316, 33 16, 33 16 8 16 16, 68 16, 68 68 9 58 16, 18 16, 18 58 16, 18 1016 56 56 16 56 11 16 56, 58 56, 58 16 56, 58 12 16 56 56 16 56 13 35 6868 35 14 16 16, 69 16, 69 69 15 35, 52 52 52 35 52 16 16 18, 56 18, 5616 18, 52 17 35 52 52 35 52 18 35 51 51 35 51 19 58 40 40 58 40 20 59 5252 59 52 21 59 16 16 59 16 22 35 16, 18 16, 18 35 16, 18 23 35 16, 1816, 18 35 16, 18 24 35 43 43 35 43 25 16 52 52 16 52 26 35, 16 35 35 1627 58 31, 39, 58 31, 39, 58 31, 39 28 31, 59 31 31 59 31 29 31, 16 31 3116, 35 31 30 35, 16 31 31 16, 35 31 31 35, 58 35, 58 35, 58 32 35, 33 3333 35 33

[0141] TABLE 5B 33 16, 35 16 16 35 34 52, 40 52, 66 52, 66 40 66 35 5866, 58 66, 58 66 36 16, 56 16 16 56 37 58, 34 58 58 34 38 16 16, 43 16,43 43 39 31, 35 31 31 35 40 33, 16 33 33 16 41 16 18 18 16 42 16, 40 negneg 16, 40 43 16, 56 neg neg 16, 56 44 16, 59 16 16 59 45 35 35, 66 35,66 66 46 16, 40 16 16 40 47 34, 58 58 58 34 48 6, 18 6, 18, 69 6, 18, 6969 49 58 51 51 58 51 50 59 11 11 59 11

[0142] The HPV genotyping kit of the invention is an implement that candetect HPV infection in a simple and accurate manner, as well asidentify the types of infecting HPV, and therefore it may contribute toearly diagnosis, prevention, and treatment of cervical cancer.

[0143] While the present invention has been described in detail withreference to the preferred embodiments, those skilled in the art willappreciate that various modifications and substitutions can be madethereto without departing from the spirit and scope of the presentinvention as set forth in the appended claims.

1 54 1 30 DNA Artificial Sequence porbe HPV 13 1 acatcatctc tttcagacacatataaggcc 30 2 30 DNA Artificial Sequence probe HPV 26 2 agtacattatctgcagcatc tgcatccact 30 3 30 DNA Artificial Sequence probe HPV 30 3ttatccacat ataattcaag ccaaattaaa 30 4 30 DNA Artificial Sequence probeHPV 43 4 cctctactga ccctactgtg cccagtacat 30 5 32 DNA ArtificialSequence porbe HPV 53 5 tctacatata attcaaagca aattaaacag ta 32 6 31 DNAArtificial Sequence probe HPV 54 6 tacagcatcc acgcaggata gctttaataa t 317 30 DNA Artificial Sequence probe HPV 54 7 catccacgca ggatagctttaataattctg 30 8 33 DNA Artificial Sequence probe HPV 55 8 tgtgctgctacaactcagtc tccatctaca aca 33 9 33 DNA Artificial Sequence probe HPV 57 9gaaactaatt ataaagcctc caattataag gaa 33 10 31 DNA Artificial Sequenceprobe HPV 61 10 ctgtatctga atataaagcc acaagcttta g 31 11 30 DNAArtificial Sequence probe HPV 61 11 cctgtatctg aatataaagc cacaagcttt 3012 30 DNA Artificial Sequence probe HPV 62 12 cctccactgc tgcagcagaatacacggcta 30 13 33 DNA Artificial Sequence probe HPV 64 13 tacaaatccaccatatgcaa acactaattt taa 33 14 30 DNA Artificial Sequence probe HPV 6714 tatgttctga ggaaaaatca gaggctacat 30 15 33 DNA Artificial Sequenceprobe HPV 68 15 tttgtctact actactgaat cagctgtacc aaa 33 16 33 DNAArtificial Sequence probe HPV 69 16 aatctgcatc tgccactttt aaaccatcag att33 17 30 DNA Artificial Sequence probe HPV 70 17 aacggccata cctgctgtatatagccctac 30 18 30 DNA Artificial Sequence probe HPV 70 18 accgaaacggccatacctgc tgtatatagc 30 19 30 DNA Artificial Sequence probe HPV 74 19cgccttctgc tacatataat agttcagact 30 20 30 DNA Artificial Sequence probeHPV JC9710 20 aaacaccctc tgacacatac aaggcttcca 30 21 30 DNA ArtificialSequence probe HPV 16 21 gtgctgccat atctacttca gaaactacat 30 22 34 DNAArtificial Sequence probe HPV 16 22 tatgtgctgc catatctact tcagaaactacata 34 23 30 DNA Artificial Sequence probe HPV 34 23 ggtacacaatccacaagtac aactgcacca 30 24 30 DNA Artificial Sequence probe HPV 35 24ttctgctgtg tcttctagtg acagtacata 30 25 36 DNA Artificial Sequence probeHPV 35 25 gcacggtctg tgtgttctgc tgtgtcttct acgtgc 36 26 30 DNAArtificial Sequence probe HPV 40 26 cttatgtgct gccacacagt cccccacacc 3027 30 DNA Artificial Sequence probe HPV 56 27 tattagtact gctacagaacagttaagtaa 30 28 30 DNA Artificial Sequence probe HPV 58 28 cactgaagtaactaaggaag gtacatataa 30 29 30 DNA Artificial Sequence probe HPV 59 29tctactactt cttctattcc taatgtatac 30 30 30 DNA Artificial Sequence probeHPV 66 30 ctaaaagcac attaactaaa tatgatgccc 30 31 30 DNA ArtificialSequence probe HPV 6 31 atccgtaact acatcttcca catacaccaa 30 32 30 DNAArtificial Sequence probe HPV 11 32 atctgtgtct aaatctgcta catacactaa 3033 30 DNA Artificial Sequence probe HPV 18 33 tgcttctaca cagtctcctgtacctgggca 30 34 30 DNA Artificial Sequence probe HPV 31 34 tgtttgtgctgcaattgcaa acagtgatac 30 35 30 DNA Artificial Sequence probe HPV 33 35tttatgcaca caagtaacta gtgacagtac 30 36 30 DNA Artificial Sequence probeHPV 39 36 tctacctcta tagagtcttc cataccttct 30 37 30 DNA ArtificialSequence probe HPV 42 37 ctgcaacatc tggtgataca tatacagctg 30 38 30 DNAArtificial Sequence probe HPV 44 38 gccactacac agtcccctcc gtctacatat 3039 30 DNA Artificial Sequence probe HPV 45 39 acacaaaatc ctgtgccaagtacatatgac 30 40 30 DNA Artificial Sequence probe HPV 51 40 agcactgccactgctgcggt ttccccaaca 30 41 30 DNA Artificial Sequence probe HPV 52 41tgctgaggtt aaaaaggaaa gcacatataa 30 42 30 DNA Artificial Sequenceb-globin 42 tgcacctgac tcctgaggag aagtctgccg 30 43 23 DNA ArtificialSequence primer Gp5+ 43 tttkttachg tkgtdgatac yac 23 44 25 DNAArtificial Sequence primer Gp6+ 44 gaaahataaa ytgyaadtca taytc 25 45 18DNA Artificial Sequence primer BG 1 45 atacaagtca gggcagag 18 46 20 DNAArtificial Sequence primer BG 2 46 cttaaacctg tcttgtaacc 20 47 30 DNAArtificial Sequence probe HPV 16 47 gtcattatgt gctgccatat ctacttcaga 3048 30 DNA Artificial Sequence probe HPV 34 48 tacacaatcc acaagtacaaatgcaccata 30 49 30 DNA Artificial Sequence probe HPV 35 49 gtctgtgtgttctgctgtgt cttctagtga 30 50 30 DNA Artificial Sequence probe HPV 40 50gctgccacac agtcccccac accaacccca 30 51 30 DNA Artificial Sequence probeHPV 56 51 gtactgctac agaacagtta agtaaatatg 30 52 30 DNA ArtificialSequence probe HPV 58 52 attatgcact gaagtaacta aggaaggtac 30 53 30 DNAArtificial Sequence probe HPV 59 53 ctgtgtgtgc ttctactact gcttctattc 3054 30 DNA Artificial Sequence probe HPV 66 54 ctattaatgc agctaaaagcacattaacta 30

What is claimed is:
 1. A probe comprising a nucleotide sequence whichcan complementarily bind to DNA of Human Papillomavirus (HPV) and isselected from the group consisting of oligonucleotides having nucleotidesequences set forth in SEQ ID. NO: 1, SEQ ID NO: 3 to 28, and SEQ ID NO:30, and oligonucleotides having nucleotide sequences complementary tothe said oligonucleotide.
 2. A DNA chip for genotyping HPV comprising atleast one HPV probe selected from the group consisting of probesaccording to claim
 1. 3. The DNA chip according to claim 2, wherein theDNA chip further comprises one or more HPV probes selected from thegroup consisting of probes having nucleotide sequences set forth in SEQID NO: 2, SEQ ID NO: 29, and SEQ ID NO: 31 to 41 and nucleotidesequences complementary to the said nucleotide sequences.
 4. The DNAchip according to claim 3, wherein the DNA chip comprises: (i) one ormore HPV probes selected from the group consisting of probes havingnucleotide sequences set forth in SEQ ID NO: 1 to 20, and nucleotidesequences complementary to the said nucleotide sequences; (ii) 8 HPVprobes selected from the group consisting of probes having nucleotidesequences set forth in SEQ ID NO 21 to 3 and nucleotide sequencescomplementary to the said nucleotide sequences; and (iii) 11 HPV probeshaving nucleotide sequences set forth in SEQ ID NO. 31 to 41 andnucleotide sequences complementary to the said nucleotide sequences. 5.The DNA chip according to claim 4, wherein the DNA chip comprises: (i) 9HPV probes selected from the group consisting of probes havingnucleotide sequences set forth in SEQ ID NO: 1 to 20; (ii) 8 HPV probeshaving nucleotide sequences set forth in SEQ ID NO: 21 to 30 andnucleotide sequences complementary to the said nucleotide sequences; and(iii) 11 HPV probes having nucleotide sequences set forth in SEQ ID NO:31 to 41 and nucleotide sequences complementary to the said nucleotidesequences.
 6. The DNA chip according to claim 5, wherein the DNA chipcomprises: (i) 3 HPV probes having nucleotide sequences set forth in SEQID NO: 4, 15, and 16; (ii) 8 HPV probes selected from the groupconsisting of probes having nucleotide sequences set forth in SEQ ID NO:21 to 30 and nucleotide sequences complementary to the said nucleotidesequences; and (iii) 11 HPV probes having nucleotide sequences set forthSEQ ID NO. 31 to 41 and nucleotide sequences complementary to the saidnucleotide sequences.
 7. An HPV genotyping kit which comprises: (i) aDNA chip for HPV genotyping according to any one of claims 2 to 6; (ii)primers for amplifying sample DNA by PCR; and (iii) means for labelingamplified DNA hybridized with the said DNA chip.
 8. The HPV genotypingkit according to claim 7, wherein the DNA chip further comprisesposition markers to locate probes.
 9. The HPV genotyping kit accordingto claim 8, wherein the position markers are selected from the groupconsisting of β-globin, actin, and glyceraldehyde-3-phosphatedehydrogenase gene.
 10. The HPV genotyping kit according to claim 7,wherein the means for labeling are selected from the group consisting ofCy5, Cy3, biotin-binding material, EDANS(5-(2′-aminoethyl)amino-1-naphthalene sulfuric acid),tetramethylrhodamine (TMR), tetramethylrhodamine isocyanate (TMRITC),x-rhodamine, and Texas red.
 11. The HPV genotyping kit according toclaim 7, wherein the amplifying of sample DNA by PCR is two-stepamplification in which a chain-extension step, that is, the firstamplification step and the second amplification step, are carried outfor 20-30 seconds and 10-15 seconds, respectively.
 12. The HPVgenotyping kit according to claim 9, wherein the amplifying of sampleDNA by PCR is carried out by simultaneously adding primers foramplifying HPV and primers for amplifying β-globin.
 13. The HPVgenotyping kit according to claim 7, wherein the primers compriseprimers for amplifying β-globin which has a nucleotide sequence setforth in SEQ ID NO. 45 or
 46. 14. A primer for amplifying β-globin whichhas a nucleotide sequence set forth in SEQ ID NO: 45 or 46.